Moringa leaf extract and green algae improve the growth and physiological attributes of Mentha species under salt stress

Climate change, food scarcity, salt stress, and a rapidly growing population are just a few of the major global challenges. The current study examined into whether Moringa oleifera (L.) leaf extract and green algae (Ulva intestinalis) could help improve salt tolerance in Mentha species (Mentha piperita; Mentha longifolia). Moringa leaf extract (MLE) and green algae (GA) were applied to Mentha seedlings under three different salt treatments: 0 mM, 20 mM, 40 mM, 60 mM, and 90 mM, respectively. For each treatment, three biological replicates were conducted, with each replicate containing at least three plants. Mentha species were negatively affected by salt stress in terms of shoot length, fresh and dry weight, photosynthetic pigments, and antioxidant enzyme activities. However, the use of MLE and GA significantly improved the development and physiology of Mentha species under salt stress conditions. The MLE and GA treatments dramatically (p ≤ 0.001) increased SOD activity by 7% and 10%, CAT activity by 16% and 30%, APX activity by 34% and 56%, GPX activity by 12% and 47%, respectively, in Mentha piperita seedlings, which in turn strikingly increased superoxide dismutase (SOD) activity by 6% and 9%, catalase (CAT) activity by 15%, 28% and 44%, 27%, ascorbate peroxidase (APX) activity by 39% and 60%, glutathione peroxidase (GPX) activity by 23% and 58%, respectively, in Mentha longifolia seedlings, relative to the control. Aiming to answer questions about the relationship between plant extraction and traditional agricultural methods, this research greatly advances the goal of sustainable development for improving plant productivity by providing a much safer and more environmentally friendly adaptability.

www.nature.com/scientificreports/ crops [20][21][22][23][24][25] . Ulva intestinalis L. is a marine green alga in the Ulvaceae family with a tubular frond and unbranched thalli 26 . It is a rich source of physiologically active molecules such as essential fatty acids, vitamins, amino acids, minerals, and growth stimulating substances; they have also been found to boost plant growth performance, antioxidant activities, and tolerance to abiotic stress 27,28 .
Mentha species are members of the Lamiaceae family, which possesses medicinal and fragrant properties. Since this particular species displays significant biological activities, it has been utilized as a treatment for a variety of respiratory conditions, including bronchitis, sinusitis, and even the common cold 29 . Moreover, it has the potential to be employed in the pharmaceutical and food industries as an efficient and cost-effective source of natural commercial antioxidants 29 . However, no research has been undertaken to our knowledge on the influence of MLE and GA extracts on the growth and physiology of Mentha species under salt stress conditions. Thus, the primary goal of this study is to investigate into the potential effects of MLE and GA on the growth and physiological attributes of Mentha species grown under salt stress conditions. The findings of this study will aid in improving Mentha species productivity in salt-stressed conditions.

Materials and methods
Experimental particulars. The Department of Biology, College of Science, Imam Abdulrahman Bin Faisal University (26.3928° N, 50.1926° E) undertook this study to investigate the effect of MLE and GA on the growth and physiology of Mentha species (Mentha piperita L. and Mentha longifolia L.) identified by Šarić-Kundalić et al. 30 , and growing under salt stress. Cultivated (Ulva intestinalis L.) identified based on Budd 31 techniques, and collected from Az Zakhnuniyah is an island located on the western coast of the Arabian Gulf (N 25° 54′ 72.94″, E 50° 32′ 53.31″) and Moringa (Moringa oliefera L.) leaves were collected from Al-Ahsa city market (Voucher number-IAU:104598). On the other hand, Mentha seeds were collected from the local market in Dammam, Saudi Arabia. The experiment used a completely randomised design with split plot layouts. Pots (40 cm in height and 25 cm diameter) were filled with compost, sand (45.29%), silt (36.22%), and clay (21.14%), with pH and EC of 7.6 and 2.52 dS m −1 , respectively. Soil pH was measured by pH meter (Divinext 3), whereas the EC was measured by EC meter (HI98331). In each of the pots, three seeds of each Mentha species were sowed. This study was performed with the local (Saudi Arabia) regulations implemented for studying towards the plants.
Salt stress treatments and preparation of extracts. Treatments were prepared based on the methods of Gholamnia et al. 8 . During the experiment, different doses of NaCl (0, 20, 40, 60, and 90 mM) were added to the experimental pots to produce salt stress. Moringa leaves that were mature and healthy were harvested and cleaned with tap water before being stored in the refrigerator overnight. An assembled machine was used for the extraction procedure. Distilled water was used to dilute the extracts to a concentration of 3%. To eliminate pollutants, tap water and distilled water were used to rinse Ulva intestinalis. It was homogenized in distilled water (1:1 by volume) at room temperature and stored until further use was needed. 100% of the liquid extract was consumed. The final extract yielded a 2% solution in distilled water.
Determination of growth parameters. Plant lengths determined by using a metric scale and expressed in centimeter (cm). The plant materials were split into shoots and roots after being cleaned with double distilled water to eliminate sand particles. The fresh weights (FW) and dry weights (DW) were measured with an analytical balance (HR-200) and expressed in grams (g).
Photosynthetic pigment determination. Arnon 32 approach was used to extract photosynthetic pigments. At room temperature, a 0.25 g leaf sample was taken and ground with 5 ml of 80% acetone. After that, the extract was centrifuged at 3000 rpm for 10 min at 40 °C. The absorbance of the supernatant at 663 and 645 nm was used to determine the chlorophyll a and b concentrations.
Proline determination. The Bates et al. 33 method was used to estimate proline concentration. 10 mL of aqueous sulfosalicylic acid and 0.5 g of newly plucked leaves (3%). After that, the mixture was filtered through a Whatman No. 40 filter paper. The mixture was placed in test tubes, and 2 mL of ninhydrin solution and 2 mL of glacial acetic acid were added. The mixture was then heated at 95 °C for over an hour before being placed in an ice bath to cool. The mixture was then extracted with 10 mL of toluene as a chromophore, and the reaction mixture was constantly circulated via an air stream for 1-2 min to separate the aqueous phase from the chromophore, which contained toluene. Finally, the separated colored phase was allowed to dry at room temperature for 2-3 min before its absorbance was measured with a spectrophotometer to be 520 nm.
Total sugar content determination. The method described by Du Bois et al. 34 was used to calculate the total soluble sugar content (1956). To extract 0.5 g of fresh leaves, 10 mL of ethanol (80%) was employed. After centrifugation, the supernatant was combined with 2.5 mL of 5% phenol solution (v/v) and 0.5 mL of sulfuric acid. To heat the combination, it was immersed in a water bath for 20 min. A standard curve was used to calculate the total soluble sugar concentration, and the absorbance at 490 nm was calculated.
Extraction and measurement of antioxidant enzyme activity. The antioxidant enzymes were extracted using the Mukherjee and Choudhuri 35 approach. In 10 mL of phosphate buffer, 0.5 g of fresh leaves were extracted (pH 7). After that, the homogenate was centrifuged at 15,000 rpm for 10 min at 4 °C. The supernatant was then maintained at 20 °C to assess antioxidant enzyme activity. www.nature.com/scientificreports/ Superoxide dismutase (SOD) activity determination. The nitro-blue-tetrazolium (NBT) reduction procedure was used to measure SOD activity 36  Statistical evaluation. The MINITAB-17 statistical software was used to perform analysis of variance (ANOVA) on the data, and the results were displayed as treatment mean ± SE (n = 3). The LSD test reveals that bars with the same letter are not statistically different at the p < 0.05 level.

Soluble sugar content.
Salt treatments of 20, 40, 60, and 90 mg/L raised the soluble sugar content in Mentha piperita by 6%; Mentha longifolia by 4%; Mentha longifolia by 18%; and Mentha longifolia by 32% compared to those in control seedlings that did not receive MLE and GE, respectively (Fig. 2). In spite of this, the exogenous infusion of MLE and GA considerably (p < 0.001) reduced the soluble sugar content.

Discussion
According to the current report results, salt stress significantly reduced the shoot and root biomass of both Mentha seedlings. The decrease in growth caused by salinity could be attributed to decreased nutrient uptake by plants or increased sodium redistribution from roots to shoots 40 . However, the current study found that applying MLE and GA to Mentha species increased their growth and physiological greatly. Similar outcome was observed in rice where MLE increased the growth and biomass under drought stress 41 . These findings suggest that MLE and GA promote Mentha species growth by altering physiological processes. In order to determine the level of salt stress, photosynthetic systems can be employed as indicators [42][43][44] . Reduced photosynthetic pigments are caused by salt stress and chlorophyll content was reported to be greater in stress-free conditions than in salt-stressed environments. In this present study, salt stress lowered the photosynthetic pigments of Mentha species. These findings back up the findings of Ahanger et al. 45 , who found that salt stress reduced chlorophyll concentration in wheat. In the current study, exogenous administration of MLE and GA significantly boosted the amount of photosynthetic pigments during salt stress. Moringa leaves are abundant in chlorophyll and carotenoids (xanthin, beta-carotene, alpha-carotene, and lutein), which have antioxidant effects 15 . MLE has also been shown to accelerate the synthesis of cytokinin's, preventing early leaf www.nature.com/scientificreports/ senescence and resulting in a bigger leaf area with higher chlorophyll content 46 . The current study findings are consistent with Khan et al. 41 discovery that MLE application significantly boosted photosynthetic pigments in wheat cultivated under favorable conditions. According to Yasmeen et al. 46 , foliar application of MLE during the tillering and heading phases increases chlorophyll a and b levels in wheat. The aqueous extract of Ulva intestinalis also increased the levels of chlorophyll a and b in parsley seedlings 47 . The total soluble sugars and proline content were determined to understand more about MLE and GA effects on salt stressed seedlings. Total soluble sugars are well-known as one of the essential organic solutes that maintain cell homeostasis [48][49][50] , and proline aids in cell osmotic adjustment in the presence of salt stress 49,50 . According to our findings, total soluble sugars and proline levels increased in the Mentha species under salt stress when compared to the control condition. A similar study in chickpea found that salt stress boosted the synthesis of total soluble sugars and proline levels in wheat 49,51 . MLE and GA combined application reduced total soluble sugars and proline levels under salt stress. Seedlings of Mentha may be able to tolerate salt stress by lowering endogenous proline production. Similarly, when exposed to salt stress alone, MLE reduced the proline concentration in Brassica napus leaves 52 . Ibrahim et al. 53 reported that ascorbic acid, betaine, glutathione, and proline are some of the bioactive components found in Ulva lactuca extract. These components, along with others, have the potential to alleviate the negative effects of salt stress.
Antioxidant defenses are essential in determining a plant's tolerance for stressful conditions [54][55][56][57][58] . With the beginning of salt stress, the activities of enzymatic antioxidants were found to be increased in the Mentha seedlings. Hanafy 59 found a significant increase in the activities of enzyme antioxidants (GR, SOD, APX, and GPX) in rice that had been exposed to salt stress. The use of MLE and GA increased the antioxidant activity of enzymatic antioxidants in Mentha species, which was especially noticeable under salt stress. Increased SOD,   www.nature.com/scientificreports/ CAT, APX, and GPX activity may be related with the activation of antioxidant responses that protect the plant from oxidative damage, according to our findings. According to Foyer and Noctor 60 , the initiation of enzymatic antioxidant activities in plants is a natural response for resisting oxidative stress. Similarly, MLE administration resulted in a significant increase in SOD activity in soybean, which was followed by the application of glutathione reductase (GR) and APX, respectively. Zaki and Rady 61 found that seed soaking or foliar spray treatment of MLE increased the antioxidant enzyme activities such as SOD, and APX in common bean (Phaseolus vulgaris L.) plants. Microalgae, on the other hand, were found to boost SOD, CAT, APX, and peroxidase (POD) activities in wheat seedlings under salt stress 62 . Furthermore, similar studies were conducted on several plants and showed that using Ulva lactuca and marine algae extracts increased the antioxidant enzyme activities. The increase in enzyme activity could be indicative of the presence of antioxidant and osmoprotectant substances.

Conclusion
Salt stress has a deleterious impact on the growth and physiology of the Mentha species. MLE and GA demonstrated the best biostimulant potential in terms of improved growth and physiology of Mentha seedlings grown under normal and salt stress. Foliar application of MLE and GA significantly improved photosynthetic pigments, osmolytes, and antioxidant enzyme activity under normal and salt stress conditions. Overall, these findings suggest that MLE and GA can be used to promote field plant development in both normal and salt-stressed environments. More research is required, however, to determine the effectiveness of MLE and GA in reducing the harmful effects of soil salinization on plants, as well as the optimal dose. Furthermore, the molecular processes underlying MLE and GA-mediated salt tolerance in plants must be understood.